Vaccine-induced protection against SARS-CoV-2 requires IFN-γ-driven cellular immune response

The overall success of worldwide mass vaccination in limiting the negative effect of the COVID-19 pandemics is inevitable, however, recent SARS-CoV-2 variants of concern, especially Omicron and its sub-lineages, efficiently evade humoral immunity mounted upon vaccination or previous infection. Thus, it is an important question whether these variants, or vaccines against them, induce anti-viral cellular immunity. Here we show that the mRNA vaccine BNT162b2 induces robust protective immunity in K18-hACE2 transgenic B-cell deficient (μMT) mice. We further demonstrate that the protection is attributed to cellular immunity depending on robust IFN-γ production. Viral challenge with SARS-CoV-2 Omicron BA.1 and BA.5.2 sub-variants induce boosted cellular responses in vaccinated μMT mice, which highlights the significance of cellular immunity against the ever-emerging SARS-CoV-2 variants evading antibody-mediated immunity. Our work, by providing evidence that BNT162b2 can induce significant protective immunity in mice that are unable to produce antibodies, thus highlights the importance of cellular immunity in the protection against SARS-CoV-2.

We report here that BNT162b2 vaccination confers protective immunity against both Alpha, Omicron BA.1 and BA.5.2 infection in both μMT mouse models. The protection is associated with lower viral replication, reduced pulmonary pathology, and elevated IFN-γ production in blood, nasal turbinate and lung tissue of vaccinated μMT mice. IFN-γ depleting antibody significantly increases Omicron BA.1 replication in vaccinated μMT mice but not that of vaccinated WT mice, suggesting the IFN-γ signaling is a core component of the protective immunity in the vaccinated μMT mice. We further demonstrate that both CD4 + and CD8 + T cells contributed to the vaccine-induced protective immunity against SARS-CoV-2 in nasal turbinate and lung tissue from μMT mice. Together, our data suggest that T cell-mediated immunity provides significant immunity in the absence of antibody-mediated immunity in vaccinated μMT mice upon SARS-CoV-2 infection and retains the ability to respond to emerging variants of concern of SARS-CoV-2.

BNT162b2 induces robust cellular responses in B lymphocytedeficient mice
To evaluate the cellular responses in μMT mice, we collected spleen tissues from vaccinated and unvaccinated C57BL/6 J wild type (WT) and μMT mice. The splenocytes were then stimulated with spike protein of SARS-CoV-2 (Fig. 1a). We first quantified antibodies titer against SARS-CoV-2 spike with ELISA and confirmed that antibody response was absent in BNT162b2-vaccinated μMT mice (Fig. 1b). Our flow cytometry analysis demonstrated that 13.2% of CD4 + and 18.5% of CD8 + T memory cells in spleen tissue of μMT mice immunized with BNT162b2 secreted IFN-γ upon stimulating with spike proteins of Alpha variant (Fig. 1c). In parallel, spike protein of Omicron BA.1 induced IFN-γ production in 17.3% of CD4 + and 16.1% of CD8 + T memory cells, suggesting robust induction of cellular responses upon stimulation of spike proteins from different SARS-CoV-2 variants (Fig. 1d). Importantly, the mean percentage of CD4 + and CD8 + T memory cells   b Antibodies titer specific for spike protein of SARS-CoV-2 was measured by ELISA (n = 5). c-f The splenocytes were stimulated by spike proteins of SARS-CoV-2 Alpha or Omicron BA.1 variant and subjected to flow cytometry analysis for IFN-γ and IL-4 responses (n = 3). Data are presented as mean ± SD. Statistical significance was calculated using one-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant). Figure 1a was created with BioRender.com.
producing IFN-γ is higher in vaccinated μMT mice than in WT mice (Fig. 1c, d), suggesting a higher level of activation of cellular responses in the vaccinated μMT mice when compared to WT mice. In contrast, IL-4 expression, which reflects the differentiation of B cells into plasma cells 13 , was not stimulated by the spike proteins in either WT or μMT mice (Fig. 1e, f). Besides, it is reported that CD8 + T cells in B celldeficient mice were skewed more toward effector phenotype during viral infection 14,15 . Interestingly, our data revealed that stimulation of spike protein of SARS-CoV-2 could also induce the skewed differentiation of CD8 + T cells into highly cytotoxic terminal effector cells in the spleen of vaccinated µMT mice (Fig. S1a). The proportion of longlived CD8 + T memory cell precursors is, however, dramatically reduced in vaccinated µMT mice compared to that in vaccinated WT mice (Fig.  S1a). Taken together, our results demonstrated that robust cellular immunity was elicited in μMT mice immunized with BNT162b2 in the absence of humoral immunity.
SARS-CoV-2 pathogenesis is associated with the accumulation of proinflammatory myeloid cells within the lung tissue in mouse model 16,17 . To identify correlates of vaccine-induced immunity in C57BL/6 J mice, we assessed the histopathological changes in the lung tissues of vaccinated and unvaccinated mice after SARS-CoV-2 Alpha and Omicron BA.1 challenge. For mice infected with Alpha, lungs in vaccinated WT mice had the least severe pulmonary pathology with relatively intact structure at 2 dpi or 4 dpi (Fig. 2d). More interstitial pneumonia and immune cell influx were observed in unvaccinated WT mice. Similarly, reduced in vivo pathology was detected in vaccinated μMT mice when compared with the unvaccinated ones. For mice infected with Omicron BA.1, the pathology of virus-induced lung tissues was milder than that of counterparts infected with Alpha, in keeping with recent findings 18 (Fig. 2d, e). In addition, the degree of inflammatory cell infiltration in the lungs of vaccinated μMT mice was lower than that observed in the lungs of un-vaccinated μMT mice (Fig. 2d, e). Together, these findings indicated that BNT162b2 vaccination is capable of inducing protective immunity in μMT mice in the absence of the antibody response, suggesting cellular immunity may serve to protect against SARS-CoV-2 infection.
Cytokines are key coordinators of the cellular immune response and essential to the activation of T cells. We next evaluated the impact of BNT162b2 vaccination on the cytokine milieu in mice infected with SARS-CoV-2 Alpha (Fig. 3a, b). Cytokine profiling of blood samples from mice challenged with Alpha revealed that IFN-γ, CXCL1, MCP-1 and IP-10 protein concentrations were significantly higher in vaccinated μMT mice than those in the unvaccinated mice (Fig. 3c). In particular, Alpha challenge drove a 253-fold (p < 0.0001) increase of IFN-γ secretion in vaccinated μMT mice when compared to unvaccinated μMT mice (Fig. 3c), suggesting strong activation of the cellular immunity upon viral challenge in vaccinated μMT mice. On the contrary, BNT162b2 vaccination is associated with a marked decrease of these key cytokines in WT mice when compared with unvaccinated ones (Fig. 3c).
We next measured the cytokine levels in lung and NT tissue of vaccinated and unvaccinated mice upon Alpha or Omicron BA.1 infection by quantitative reverse transcription PCR (RT-qPCR). In line with the data in blood samples, BNT162b2 vaccination increased the expression of IFN-γ, MCP-1, and IP-10 in lung and NT tissues of μMT mice challenged with Alpha or Omicron BA.1 ( Fig. 3d-i).
The K18-hACE2 mouse model has been frequently used to assess SARS-CoV-2 pathogenesis 12,19 . To evaluate the impact of B cell depletion in the K18-hACE2 mice, we obtained K18-hACE2 μMT mice by breeding K18-hACE2 mice with μMT mice. In the K18-hACE2/μMT mice, BNT162b2 vaccination similarly offered protection against Alpha and Omicron BA.1 infection. Alpha and Omicron BA.1 replication in the lung and NT tissues were lower in vaccinated K18-hACE2/μMT mice when compared with un-vaccinated ones (Fig. 4b, c). In line with the results in C57BL/6 J model, we observed ameliorated lung pathology in vaccinated WT or μMT mice when compared with unvaccinated ones (Fig. 4d, e). For cytokine profiling, we quantified IFN-γ, MCP-1, and IP-10 expression in lung tissue and NT in the mice by RT-qPCR. We found that BNT162b2 vaccination increased the expression of IFN-γ, MCP-1, and IP-10 in lung and NT tissues of K18-hACE2 μMT mice challenged with Alpha or Omicron BA.1 (Fig. 4f-k), similar to what we observed in C57BL/6 J μMT mice ( Fig. 3d-i). Taken together, these findings indicate that BNT162b2 elicits significant protective immunity against SARS-CoV-2 Alpha and Omicron BA.1 in B lymphocyte-deficient mice.
T cell-mediated immunity is highly activated in B lymphocytedeficient mice upon SARS-CoV-2 infection To characterize the vaccine-induced protective immunity in μMT mice, we performed bulk RNA-seq of WT and µMT C57BL/6 J mice lung and NT tissues with or without vaccination on 2 dpi (Fig. 5a). Gene functional enrichment analysis based on the significantly differential expressed genes showed prominent immune characters between different conditions (Fig. 5b). We found that BNT162b2 vaccination boosted both humoral and cellular immunity in both NT and lung tissues in WT mice (Fig. 5b). In contrast, due to the lack of mature B cells, only genes involved in T cell activation and T cell receptor signaling pathways were significantly upregulated in vaccinated µMT mice comparing to unvaccinated µMT mice, suggesting that cellular immunity can be effectively activated by BNT162b2 vaccination in B cell-deficient mice (Fig. 5b). Cell type functional enrichment also indicated that the function of CD8 + T cell is highly enriched in vaccinated µMT mice when compared to the unvaccinated µMT mice (Fig.  S2). According to the differential gene expression analysis, we found that T cell-related genes/pathways were significantly upregulated in the vaccinated µMT mice in both NT and lung tissue when compared to unvaccinated µMT mice (Fig. 5c, d). Among these genes, integral membrane glycoprotein CD8a serves as a coreceptor of class I MHC molecular in CD8 + T cells, and cell adhesion molecular CD6 that regulates T cell responses was both upregulated in NT and lung tissues ( Fig. 5c, d). Besides, the cxcr6 gene, which controls the localization of resident memory T lymphocytes to different compartments of the lung and maintains airway resident memory T lymphocytes 20 , was also significantly upregulated in vaccinated µMT mice (Fig. 5c, d).

IFN-γ facilitates vaccine-induced immunity in B lymphocytedeficient mice
As mentioned earlier, IFN-γ secretion was substantially upregulated in serum, NT, and lung tissues of vaccinated μMT mice. To further evaluate the role of IFN-γ in the vaccine-induced protective immunity in μMT mice, we injected IFN-γ depleting antibody into vaccinated WT and μMT mice before Omicron BA.1 challenge (Fig. 6a). Our results demonstrated that the IFN-γ depleting antibody significantly increased Omicron BA.1 replication by 19.3-fold (P = 0.0029) in the lung of vaccinated μMT mice. In contrast, the IFN-γ depleting antibody did not modulate Omicron BA.1 replication in the lungs of vaccinated WT mice (0.7-fold, P = NS) (Fig. 6b). In the NT, the IFN-γ depleting antibody has a trend to promote virus replication in vaccinated WT and μMT mice, albeit no statistical significance was reached (Fig. 6b). In keeping with these observations, treatment of IFN-γ depleting antibody resulted in more severe lung pathology in vaccinated μMT mice while no significant change in lung pathology was observed in vaccinated WT mice when IFN-γ depleting antibody was administered (Fig. 6c). A recent study reported that supplementation of IFN-γ reverses the agedependent COVID-19 phenotype in mice 21 . Therefore, we next treated naïve μMT mice daily with 10 μg IFN-γ from −1 to 1 d.p.i. to investigate whether administration of IFN-γ could alleviate the disease severity upon SARS-CoV-2 infection. Supplementation of IFN-γ resulted in lower viral load and milder lung pathology in NT and lung tissues of naïve μMT mice than PBS control group when infected with SARS-CoV-2 Alpha (Fig. 6e, f). Together, our results indicate that IFN-γ contributes to the vaccine-induced cellular immunity that offers protection against SARS-CoV-2 in B lymphocyte-deficient mice.
BNT162b2 induces protective immunity against SARS-CoV-2 Omicron BA.5.2 in B lymphocyte-deficient mice SARS-CoV-2 Omicron BA.5, which has substantially higher immune escape capacity than BA.1, are the dominant strain in many countries as of January 2023 [22][23][24] . To test whether BNT162b2 could induce protective immunity against SARS-CoV-2 Omicron BA.5.2 in μMT mice, we infected vaccinated C57BL/6 J and K18-hACE2 mice with SARS-CoV-2 Omicron BA.5.2 and evaluated the viral replication in these mice. The quantitative RT-PCR showed that the viral load in lung tissues of vaccinated μMT mice is 34-fold (P = 0.0283) and 188-fold (P = NS) lower than C57BL/6 J and K18-hACE2 unvaccinated μMT mice at 2 d.p.i., respectively (Fig. 8b). Although much lower viral load was detected in NT of both C57BL/6 J and K18-hACE2 vaccinated mice at 2 and 4 d.p.i., the statistical difference was not seen between the vaccinated and unvaccinated groups (Fig. 8b). In line with the viral replication, the lung pathology was also alleviated in vaccinated mice (Fig. 8c). Consistent with the therapeutic effect of IFN-γ in naïve μMT mice against SARS-CoV-2 Alpha, supplementation of IFN-γ could also mitigate the viral replication and lung pathology in naïve μMT mice upon Omicron BA.5.2 challenge (Fig. 8d, e).

Discussion
A large body of evidence has emphasized the essential role of neutralizing antibodies induced by vaccines or natural infections in the protection against SARS-CoV-2. BNT162b2 vaccine has been reported to elicit an adaptive humoral and poly-specific cellular immune response in both animal models and humans [1][2][3] . In this study, we investigated whether vaccine-induced cellular immunity is sufficient to elicit protective immunity against SARS-CoV-2 by using µMT mice that lack mature B cells. A recent study has demonstrated that both humoral and cellular adaptive immunity are important in viral clearance after primary infection of SARS-CoV-2 by using AAV-hACE2 µMT mice 25 . Here, we describe the use of two different mouse models to identify components of adaptive immunity responsible for viral clearance induced by BNT162b2. We measured the viral load in both nasal turbinate (NT) and lung tissue of the vaccinated and unvaccinated mice after the viral challenge. The clearance of SARS-CoV-2 is dramatically more effective in both vaccinated C57BL/6 J WT and µMT mice when compared with unvaccinated ones, suggesting the induction of sufficient cellular immunity by BNT162b2 in mice without humoral responses. Similar results were obtained by using K-18 hACE2 mouse model, though a quicker clearance of SARS-CoV-2 was observed in C57BL/6 J mice as indicated in the previous study 11 . For survival test in K18-hACE2 model, vaccination of BNT162b2 resulted in a 20% survival rate in µMT mice as opposed to 0% in unvaccinated µMT mice in the setting of Alpha variant challenge (Fig. S3a). Interestingly, a substantially higher level of viral RNA was detected in the brain tissue of vaccinated µMT mice when compared with that in vaccinated WT mice, suggesting antigenspecific antibodies are essential to contain the viral replication in brain tissue (Fig. S3c). When vaccinated µMT mice were challenged with Omicron BA.1, no death was caused due to the attenuated virulence of Omicron BA.1 (Fig. S3b) 18 . Bulk RNA-seq analysis revealed that pathways involved in T cell activation were upregulated in vaccinated µMT mice when compared to unvaccinated ones. Moreover, we found that the gene functional enrichment profile of vaccinated WT vs. µMT mice in nasal turbinate was different from what is in lung tissue. In nasal turbinate, T cell differentiation and receptor signaling pathways are upregulated in vaccinated µMT mice. Nevertheless, SARS-CoV-2 infection induced upregulated pathways involved in hemostasis and thrombosis (platelet activation, ECM-receptor interaction, etc.) in vaccinated µMT mice in lung tissue when compared to WT mice (Fig.  S4), implying that the mechanism of vaccine-induced viral clearance in the lung might be different from that in nasal turbinate in µMT mice. It is also important to point out that antibodies can also function by opsonization and complement-mediated cytotoxicity. To investigate the role of the complement system in the clearance of SARS-CoV-2, we carried out gene set enrichment analysis (GSEA) on the gene sets related to complement pattern recognition, complement proteases, complement component, complement receptor, and complement regulator. We found that the complement activation pathways were upregulated in vaccinated WT mice when compared with unvaccinated WT mice (Fig. 5b). No significant differences in the complementrelated pathways were detected in either NT or lung tissues between vaccinated and unvaccinated μMT mice (Fig. S5).
To confirm whether T cell-mediated immunity contributes to the vaccine-induced protective immunity in µMT mice, we analyzed the cytokine/chemokine profile of the blood, NT, and lung samples from vaccinated WT and µMT mice. We demonstrated that IFN-γ secretion is highly activated in vaccinated µMT mice upon viral challenge. Particularly, a 253-fold increase of IFN-γ secretion was observed in vaccinated μMT mice compared to vaccinated WT mice. Likewise, CXCL10 (IP-10) secretion in response to IFN-γ was also significantly higher in vaccinated μMT mice. These data are consistent with our finding that stimulation of spike protein of SARS-CoV-2 induces a higher percentage of IFN-γ-secreting CD4 + and CD8 + memory T cells in vaccinated µMT mice (Fig. 1c, d). By using depleting antibodies against IFN-γ, we confirmed that IFN-γ plays an important role in viral clearance in vaccinated μMT mice but not WT mice. Upon viral infection, IFN-γ acts directly on CD8 + T cells to facilitate their differentiation into cytotoxic T lymphocytes, which secret cytokines to restrain viral replication and clear infected cells 26 . The role of IFN-γ during the infection of SARS-CoV-2 is not yet fully understood 27 . Previous studies showed that IFN-γ was present in high amounts in patients with moderate to severe COVID-19 along with other proinflammatory cytokines and was associated with cytokine storm and acute lung damage in COVID-19 patients 28,29 . In this study, we demonstrated that IFN-γ facilitated the clearance of SARS-CoV-2 without inducing lethal cytokine storm in vaccinated µMT mice. Increased IFN-γ levels in lungs of vaccinated µMT MCP-1 level was quantified by q-RTPCR using RNA samples extracted from lung and NT from BNT162b2 vaccinated and unvaccinated K18-hACE2 WT/μMT mice at 2 d.p.i. and 4 d.p.i. of Alpha (f, h, j) or Omicron BA.1 variant (g, i, k) (n = 6). Scale bar = 200 μm. Data are presented as mean ± SD. Statistical significance was calculated using one-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant). Figure 4a was created with BioRender.com. mice did not either result in more severe lung pathology. Moreover, the levels of many proinflammatory cytokines such as IL-6, IL-1β, TNFα, and other interferons were not significantly elevated in vaccinated µMT mice. On the other hand, a recent study reported that therapeutic administration of IFN-γ could reverse the disease severity upon SARS-CoV-2 infection in aged WT C57BL/6 mice 21 . In line, we showed that supplementation of IFN-γ was effective in lowering the viral load in NT and lung tissues of naïve µMT mice, confirming the pivotal role of IFN-γ in vaccine-induced viral clearance in µMT mice. However, the administration of IFN-γ in naïve μMT mice resulted in less significant reduction of viral load (3.3-fold for Alpha and 22-fold for BA.5.2, respectively) when compared with the BNT vaccination (11.5-fold for Alpha and 33.8-   fold for BA.5.2, respectively). This implies that element(s) other than IFN-γ might be involved in the vaccine-induced protection. Together, we hypothesize that BNT162b2 induced IFN-γ-mediated compensatory protective immunity in the absence of humoral immunity in µMT mice without causing any detrimental effect of IFN-γ. CD4 + and CD8 + T cells are the major sources of IFN-γ production in the adaptive immune system 30 . We showed that depletion of CD8 + T cells in vaccinated μMT mice lead to a 15.6fold increase of viral load in NT compared to PBS group, which is in line with the highly enriched function of CD8 + T cell in vaccinated µMT mice by cell type enrichment analysis from RNA-seq data (Fig. S2). However, only the depletion of both CD4 + T cells and CD8 + T cells resulted in a significant increase of the viral RNA load in NT or lung tissue from vaccinated µMT mice. Therefore, our data indicate that both CD4 + and CD8 + T cells play important roles in the vaccine-induced clearance of SARS-CoV-2 in B celldeficient mice. By using flow cytometry, we demonstrated that a significantly higher percentage of CD44 + CD69 + CD8 + and CXCR6 + lung tissue-resident memory (TRM) CD8 + T cells were found in lung tissues of vaccinated μMT mice than in unvaccinated ones upon SARS-CoV-2 challenge (Fig. 7e, S7a). It has been demonstrated that CD8 + T cells in the draining lymph nodes are one of the major producers of the circulating IFN-γ in mice after BNT162b2 vaccination 31 . Likewise, our data indicated that many more IFN-γ producing CXCR6 + CD8 + TRM cells were detected in the lung tissues of vaccinated μMT mice than unvaccinated mice upon SARS-CoV-2 infection (Fig. 7f, S7b). Taken together, we demonstrated that BNT162b2 vaccination could mount robust cellular immunity through IFN-γ-secreting T memory cells in µMT mice, promoting viral reduction. These results are consistent with the recent report that robust production of IFN-γ was observed following stimulation with spike peptide in individuals with X-linked agammaglobulinemia (XLA), who received two doses of BNT162b2 32,33 . Although XLA occurs in approximately 1 in 200,000 newborns, individuals with primary B-cell immunodeficiency (PID, 500,000 cases in the United States as of 2003, and about 50,000 cases are diagnosed each year 34 ) share the symptoms with XLA individuals. Therefore, this study will shed light on the protective efficacy of BNT162b2 in these immunocompromised individuals.

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SARS-CoV-2 variants such as Omicron variants that significantly evade humoral immunity are continuously emerging. Here, we demonstrated that vaccination of BNT162b2 elicited protective immunity against Omicron BA.1 and BA.5.2 in either WT or μMT mice, although the efficacy is less significant than that against Alpha. These results are consistent with the observation that current vaccines still show robust protection against severe disease caused by Omicron and its sub-lineages despite the significantly decreased neutralizing antibody responses in the human population 35 . The protection was found to be strongly associated with vaccine-induced cellular immunity that is mediated by CD8 + T cells. Likewise, we showed that spike protein of Omicron BA.1 could stimulate the robust secretion of IFN-γ by CD8 + T cells in both WT and µMT mice vaccinated with BNT162b2 ( Fig. 1c-d).
Current COVID-19 vaccines elicit robust systemic immunity but poor immunity at the respiratory mucosa 36 . With enhanced immune evasion of the emerging Omicron variants and waning systemic immunity among the population, these vaccines such as BNT162b2 are less effective at containing viral transmission. Successful vaccineinduced mucosal immune memory within the respiratory tract is the key to preventing viral transmission. However, we did not investigate the role of vaccine-induced mucosal T cell immunity in B-cell-deficient mice in this study. Mao et al. 37 recently developed a vaccine strategy that elicited protective mucosal immunity against SARS-CoV-2 and reduced viral transmission in K18-hACE2 mice. Hence, it would be worthwhile to study if mucosal T cell immunity could reduce viral transmission in immunocompromised mouse models under the improved vaccine strategy.
A growing body of publications has reported that robust Spikespecific T cell responses were induced in BNT162b2 vaccinated patients with impaired humoral responses [4][5][6] . In this study, our data from mouse models are consistent with the above-mentioned reports in humans, demonstrating the validity of our animal model to evaluate the COVID-19 vaccine efficacy in the context of impaired humoral immunity.
We further demonstrated that T cell-mediated immunity provides significant immunity in the absence of antibody-mediated immunity in vaccinated μMT mice upon SARS-CoV-2 infection and retains the ability to respond to emerging variants of concern of SARS-CoV-2, which provide valuable insights into the clinical guidance in the vulnerable population.

Mice
This experimental protocol was approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong under CULATR 5479-20 and complied with the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (8 th edition, 2011). Six to 12-week-old mixed sex C57Bl/6 J (WT), B6.129S2-Ighmtm1Cgn/J (#002288, μMT), and B6.Cg-Tg(K18-ACE2)2Prlmn/J (#034860, K18-hACE2) were purchased from the Jackson Laboratory and were subsequently bred and housed at the SPF animal facility in the Center for Comparative Medicine Research (CCMR) of the University of Hong Kong. To obtain K18-hACE2 μMT mice, B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were cross-bred with B6.129S2-Ighmtm1Cgn/J (μMT) mice. The genotyping was conducted following protocols provided by Jackson Laboratory. Mice were euthanized by overdosing with Pentobarbital sodium (250 mg/kg) intraperitoneally.

Cell lines and viruses
Vero E6 kidney epithelial cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% sodium pyruvate and 5% fetal bovine serum (FBS) at 37°C and 5% CO 2 . 293 T cells were cultured in DMEM supplemented with 1% sodium pyruvate and 10% FBS at 37°C and 5% CO 2 . The cell line was obtained from the American Type Culture Collection (ATCC) and has been tested negative for contamination with mycoplasma. SARS-CoV-2 isolates used in the study were previously described 18,38 and were amplified in VeroE6 cells overexpressing transmembrane serine protease 2 (TMPRSS2). The VeroE6-TMPRSS2 cells were infected at a multiplicity of infection of unvaccinated) c Volcano plot showing differential expression genes from DESeq2 comparing BNT162b2 vaccinated transcriptome to unvaccinated ones in both NT and lung tissues of µMT mice. Genes with orange labels were significantly upregulated in NT or lung (Benjamini-Hochberg adjusted P value < 0.05 and gene expression fold change > 2), scatters with grey labels indicated gene upregulated in both NT and lung (Benjamini-Hochberg adjusted P value < 0.05 and gene expression fold change > 2). d Heatmap plot showing the expression levels of upregulated genes labeled in (b) across different conditions, expression of each gene was scaled in NT and lung, respectively. Figure 5a was created with BioRender.com. 0.01 for 2 to 3 days to generate a working stock, and after incubation, the supernatant was clarified by centrifugation (500 g × 5 min) and filtered through a 0.45-μm filter. To concentrate virus, filtered supernatants were applied to Amicon Ultra-15 centrifugal filter (Ultracel 100k) and spun at 1000 g for 15 min. The supernatant was then aliquoted for storage at −80°C. Viral titers were measured by standard plaque assay using Vero E6 TMPRSS2 cells. In vivo and in vitro experiments with infectious SARS-CoV-2 were performed according to the approved standard operating procedures of the Biosafety Level 3 facility at the Department of Microbiology, HKU.

Vaccination
Unused vials of Pfizer/BioNTec BNT162b2 mRNA vaccine were acquired from the Department of Health, HKSAR. Mice were anaesthetized using a mixture of ketamine (50 mg kg−1) and xylazine (5 mg kg−1), and 10 μL (1 mg) of the undiluted vaccine was injected into the left quadriceps muscle with a 32-gauge syringe.

SARS-CoV-2-specific antibody measurements
Mouse serum samples were incubated at room temperature for 30 min before use to reduce risk from any potential virus in serum. The 96-well

Histology staining
Animal tissues were collected and fixed with 10% neutral-buffered formalin. For H&E staining, tissue sections were stained with Gill's haematoxylin and eosin-Y. Images were acquired using Vectra Polaris automated quantitative pathology imaging system (Perkin Elmer). Three to four mice were sampled in each group and four to six sections from each animal were used for histology analysis.

Cytokine/chemokine quantification
Cytokines/chemokines in blood samples harvested 2 d.p.i. were assessed by using LEGENDplex™ anti-virus response panel kit (BioLegend) according to the manufacturer's instructions and quantified by using ACEA NovoCyte Quanteon flow cytometer (Agilent). The data were analyzed by LEGENDplex™ Data Analysis Software Suite. Cytokine/chemokine levels in lung or NT samples were measured by RT-qPCR with primer sets for murine IFN-γ, MCP-1 and IP-10.

RNA-seq data processing
Adapters and low-quality bases were trimmed from raw next generation sequencing data with the help of fastp (v0.20.1) using default parameters 39 . Clean data were then used for mouse and SARS-CoV-2 gene expression quantifications using Salmon (v1.6.0) respectively 40 . Mouse gene expression profile was calculated from clean data directly. SARS-CoV-2 gene expression profile was calculated from un-mapped reads which were retrieved after executing alignment to mouse reference genome by STAR (v2.7.10a) 41 . Gene different expressions analysis were carried out by DESeq2 between different condition of the same tissue. ClusterProfiler (v3.18.1) was used for immune and viral process related gene ontologies (ontologies belongs to GO:0016032 and GO:0002376) and KEGG pathway enrichment analysis, similar GO terms were merged by "simplify" function with cutoff of 0.5 42 . Cell type functional enrichment were conducted by xCell (v1.1.0) using moue gene expression profile 43 . For Gene set enrichment analysis of the complement system, gene list related to complement system were obtained from Neha et al. 44 . Ranked fold change of genes were used during the GSEA analysis with the help of R package fgsea (v1. 16.0) 45 with default parameter. Fig. 7 | CD4 + and CD8 + T cells are required for the vaccine-induced clearance of SARS-CoV-2 in NT and lung tissue in μMT mice. a Schematic diagram of vaccination, CD4 + or CD8 + T cell depletion, viral challenge, and pathological studies.
b The viral loads in the lung and NT of BNT162b2 vaccinated and unvaccinated C57BL/6 J μMT mice at 2 d.p.i. with CD4 + , CD8 + , or both T cell depletion (n = 10). (aCD4, anti-CD4 monoclonal antibody; aCD8, anti-CD8 monoclonal antibody). c Representative images of the H&E-stained lung tissues of BNT162b2 vaccinated μMT mice treated with aCD4, aCD8, both depleting antibodies or PBS. Scale bar = 200μm. d Schematic diagram of flow cytometry analysis of CD4 + and CD8 + T cells in lung tissues of vaccinated and unvaccinated C57BL/6 J μMT mice after Alpha infection. e Percentage of CXCR6 + and CD69 + CD4/8 + T cells in lung tissues of vaccinated and unvaccinated C57BL/6 J μMT mice after Alpha infection (n = 5).